and in copy number control of the
transcriptional regulator CadC in
Escherichia coli
Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
vorgelegt von
Susanne Caroline Margarethe Ude
München
1.
Gutachterin:
Prof. Dr. Kirsten Jung
Department Biologie I, Bereich Mikrobiologie, LMU München
2.
Gutachter:
Prof. Dr. Dirk Schüler
Department Biologie I, Bereich Mikrobiologie, LMU München
III
Hiermit versichere ich eidesstattlich, dass die vorliegende Dissertation von mir selbstständig und ohne unerlaubte Hilfe angefertigt wurde. Des Weiteren erkläre ich, dass ich nicht anderweitig ohne Erfolg versucht habe, eine Dissertation einzureichen oder mich der Doktorprüfung zu unterziehen. Die vorliegende Dissertation liegt weder ganz, noch in wesentlichen Teilen einer anderen Prüfungskommission vor.
München, 05. März 2013 ……….
(Susanne Ude)
Statutory Declaration:
I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources. As well I declare that I have not submitted a dissertation without success and not passed the oral exam. The present dissertation (neither the entire dissertation nor parts) has not been presented to another examination board.
München, 05. März 2013 ……….
IV
Statutory Declaration ... IV
Abbreviations ... VIII
Nomenclature ... X
Contributions ... XI
1. Introduction ... 1
1.1. Acid stress adaptation ... 1
1.2. The Cad system ... 4
1.2.1. CadA, CadB and CadC ... 4
1.2.2. Regulation of the Cad system ... 5
1.3. Steps in translation ... 7
1.4. The elongation factor EF-P ... 9
1.5. Scope of the thesis ... 11
2. Materials and Methods ... 12
2.1. Materials ... 12
2.2. Strains, plasmids and oligonucleotides ... 14
2.3 Cultivation techniques ... 28
2.3.1 Purification of YjeK ... 28
2.3.2 Lysine decarboxylase and β-galactosidase activity tests ... 28
2.3.3 Cultivations for RNA isolation ... 29
V
2.4 Molecular biological and genetic methods ... 30
2.4.1 Isolation of plasmids and genomic DNA ... 30
2.4.2 Modification of DNA ... 30
2.4.3 Polymerase chain reaction ... 30
2.4.4 Electrophoretic separation of DNA ... 31
2.4.5 DNA gel extraction and determination of nucleic acid concentration ... 31
2.4.6 DNA sequencing analysis ... 31
2.4.7 Strain construction ... 31
2.4.8 Plasmid construction ... 32
2.4.9 Preparation of competent cells and transformation ... 32
2.4.10 Isolation of RNA ... 33
2.4.11 Denaturated gel electrophoresis ... 33
2.4.12 Northern Blot analysis ... 34
2.4.13 qRT-PCR ... 34
2.5 Biochemical and analytical methods ... 35
2.5.1 Purification of YjeK and CadC ... 35
2.5.2 Preparation of CadC membrane vesicles... 35
2.5.3 Determination of the protein concentration... 35
2.5.4 SDS Page ... 36
2.5.5 Western Blot analysis ... 36
2.5.6 Lysine decarboxylase activity test ... 37
2.5.7 β-Galactosidase activity test ... 37
2.5.8 Bacterial two hybrid system ... 38
2.5.9 In vitro translation assay ... 38
VI
3.1. Importance of YjeK, YjeA and EF-P for cadBA expression ... 40
3.1.1. CadA activity in yjeK, yjeA and efp mutantsin E. coli ... 40
3.1.2. Complementation of a yjeK mutant with KamA of C. subterminale ... 44
3.2. Interaction studies ... 45
3.2.1. Dimerization of YjeK in vitro ... 45
3.2.2. Dimerization of YjeK, YjeA and EF-P with Cad components ... 46
3.3. Determining the target of EF-P in the Cad system ... 48
3.3.1. cadBA expression in yjeA, yjeK and efp mutants ... 48
3.3.1.1. Expression of lacZ under control of the cadBA promoter ... 48
3.3.1.2. cadB and cadA transcription in yjeK, yjeA and efp mutants ... 49
3.3.2. CadC as possible target of EF-P ... 51
3.3.2.1. CadC protein levels in the wildtype and yjeA, yjeK, efp mutants ... 51
3.3.2.2. cadC transcription in the wildtype and yjeA, yjeK, efp mutants ... 53
3.4. The role of EF-P in translation ... 54
3.4.1. EF-P as possible helper protein for first peptide bond formation ... 54
3.4.2. EF-P as specific translation elongation factor of CadC ... 57
3.5. Searching for the EF-P signal sequence ... 59
3.5.1. The CadC proline cluster and its role for EF-P dependency ... 59
3.5.2. The specificity of the consecutive prolines ... 62
3.5.3. Testing ribosomal stalling in vitro ... 66
3.6. EF-P and its physiological role in the Cad system ... 67
3.6.1. Comparison of E. coli and V. harveyi CadC ... 67
3.6.2. The consequence of EF-P independent CadC for cadBA expression ... 69
3.6.3. The effect of different CadC copy numbers on cadBA expression ... 71
3.7. Identification of additional EF-P dependent proteins ... 73
3.9 Proline cluster and the “plus” ... 76
VII
3.10 Differences in cadBA expression in KE and LB medium ... 81
3.11 Role of Hfq in the Cad system ... 85
4. Discussion ... 89
4.1. EF-P and its role in translation ... 89
4.2. Ribosomal stalling at prolines and additional stalling sequences ... 90
4.3. The CadC/LysP balance model ... 94
4.4. EF-P and its importance in plant and mammalian diseases ... 98
5. Summary ... 101
6. Zusammenfassung ... 103
7. Literature ... 105
Appendix ... 123
VIII
σS stationary phase specific sigma factor (rpoS)
AAA+ ATPases associated with various cellular activities
aa amino acid
ampR ampicillin resistance
AR acid resistance
ara arabinose
A-site aminoacyl-site
ASP acid shock protein
ATP adenosine-5´-triphosphate
bp base pair
cAMP cyclic adenosine-5´-monophosphate
cat chloramphenicol acetyltransferase
ddH2O double distilled water
DNA deoxyribonucleic acid
DTT dithiothreitol
EF elongation factor
E-site exiting-site
Fig. Figure
gmR gentamycin resistance
h hour (s)
GTP guanosine-5´-triphosphate
His6-tag affinity tag containing 6 histidine residues
HTH helix turn helix
IF initiation factor
kDa kilodalton
LB Lysogeny Broth
mRNA messenger ribonucleic acid
MU miller units
min minute (s)
N-formyl-methionyl-tRNAi fMet-tRNAi
IX
OL overlap
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
PDB Protein Data Bank (www.pdb.org)
pmf proton motive force
ppGpp guanosine tetraphosphate
P-site peptidyl site
PTC peptidyltransferase center
Px promoter of gene x
qRT-PCR quantitative real time PCR
rbs ribosomal binding site
RF release factor
SDS sodium dodecyl sulfate
sec second (s)
tetR tetracycline resistance
TM transmembrane domain
tRNA transfer ribonucleic acid
UV ultraviolet
v/v volume per volume
X
Gene products are numbered in a way that the first methionine of the wildtype protein is designated “1” in the amino acid sequence. N-terminal affinity tags or linker sequences are left unconsidered.
Positions of nucleotides indicate the distance from the translational start site (A of ATG is +1).
Gene deletions are depicted by the symbol ∆, gene replacements as follows: replaced gene::new gene. When only parts of the gene were deleted, the missing nucleotides are specified (for example: yjeK643-1029).
Amino acids are designated in one-letter or three-letter code. For single amino acid substitutions, the native amino acid is named in front of the corresponding amino acid
position in the protein. The substituted amino acid is terminally added (for example: CadC-P122A). The exchange of the CadC proline cluster against alanines and a serine is designated
as P/A or PPPIP/AAAIS.
Cells containing wildtype efp are termed efp+ cells. efp deletion mutants are designated as efp
-cells.
XI
Figure 11 and Figure 13:
The design for the experiments was done by Susanne Ude (SU), Dr. Jürgen Lassak (JL), Prof. Dr. Kirsten Jung (KJ). qRT-PCR experiments were done by JL to verify and complete Northern Blot results obtained by SU. Experiment for Fig. 13B was performed by SU and JL.
Table 5, Figure 14, 16, 18, 19, 20, 22, 27C:
The experiments were designed by SU, JL and KJ. The practical work was done by SU (Fig. 14, Fig. 16C, Fig. 18, Fig. 22), JL (Fig. 16B, Fig. 27C, Fig. 19, Fig. 20) and Christina Krönauer (Table 5).
Figure 21 and 25B:
The experiments were planned by SU, JL, KJ, Dr. Daniel Wilson and Dr. Agata Starosta. The practical work was done by JL (construction of pET16b-CadC templates) and Dr. Agata Starosta (in vitro translation assay).
Figure 23 and Figure 24:
Dotblots were performed by JL.
Fig. 24C: The experiment was planned by SU, qRT-PCRexperiment was performed by JL.
Table A1:
Table A1 was created by Dr. Daniel Wilson.
Experiments which are not mentioned here were made by SU.
Portions of figures, tables and text were previously published in a different form in:
Ude S., Lassak J., Starosta A.L., Kraxenberger T., Wilson D.N., Jung K. (2013):
Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.
1
1.
Introduction
1.1.
Acid stress adaptation
The interplay between humans and microbes for human health is highly important and was first described over 100 years ago by Pasteur (Schottelius, 1902). The human surfaces and the gastrointestinal tract (GI) are inhabited by approximately 1014 mutualistic microbial cells – ten times more cells than the human body consists of (Hooper & Gordon, 2001; Savage, 1977). One gram (wet weight) of the human colon contains about 1011 prokaryotes (Whitman
et al., 1998). While the gut represents an ideal milieu for bacteria as it is enriched by dietary compounds, bacteria in return help their hosts to digest polysaccharides and provide them with essential vitamins (Conly et al., 1994; Savage, 1986). As babies are born without any germs, their gut is colonized immediately after birth by first mainly facultative and later on mainly obligate anaerobic bacteria to develop a normal healthy digestive tract (Hooper, 2004; Savage, 1977). In fact, exceeding hygiene standards in early childhood can be linked to inflammatory bowel disease (IBD), represented by Crohn´s disease and ulcerative colitis, resulting in intolerance against normal gut inhabitants (Gent et al., 1994). The main colonizers consist largely of two phyla, Bacteroidetes and Firmicutes (Bäckhed et al., 2005; Eckburg et al., 2005), but also include members of the family Enterobacteriaceae. Enterobacteria can be harmless, as it is the case with most types of Escherichia coli, but they also harbor pathogens such as Salmonella typhimurium, enteropathogenic (EPEC) or enterohemorrhagic E. coli (EHEC), which disturb the normal gut flora and cause severe intestinal diseases (Stecher & Hardt, 2008).
While colonizing the gut, bacteria are exposed to substantial changes in the pH range. The stomach contains hydrochloric acid (HCl) and thereby reaches extreme low pH values of 1.5 – 2.5 (Smith, 2003). Furthermore, weak organic acids such as propionic and acetic acids, which are produced during the bacterial fermentation process, lead to additional acidic stress. These weak acids, protonated and uncharged at low pH, can freely diffuse through the cytoplasmic membrane of the bacteria and easily dissociate in the neutral cytoplasm. This leads to decreased internal pH, destabilized inactive macromolecules, and the destruction of the proton
2
et al., 1984). Consequently, bacteria are unable to keep up their proton gradient, which is crucial for ATP formation and cell metabolism. To survive these extreme stress conditions, neutrophil enterobacteria have evolved several passive and active strategies to protect their internal homeostasis (Fig. 1). Passive survival mechanisms include the impermeability of the cytoplasmic membrane for charged ions such as protons and the buffering capacity of the
cytoplasm by polyamines, proteins and nucleic acids (Slonczewski & Foster, 1996). By contrast, proton pumps actively export protons out of the cell by consumption of ATP. To
avoid further proton influx, other charged ions like potassium are imported by K+-transporters such as Trk or Kup (Bakker & Mangerich, 1981; Rhoads et al., 1976). Moreover, acidic pH can activate transcription regulators like Fur and PhoP, leading to the synthesis of diverse acid shock proteins (ASPs). These ASPs also contribute to the retention of the internal pH homestasis (Bearson et al., 1997).
Figure 1: Stress adaptation in E. coli. To survive the acidic environment of the gastrointestinal tract,
enterobacteria have evolved several different strategies: Passive mechanisms include the
impermeability of the cytoplasmic membrane for charged ions and the buffering capacity of the
cytoplasm. Proton pumps and potassium transporters belong to the active constitutive mechanisms.
Furthermore, the degradative amino acid decarboxylase systems Gad, Adi and Cad are induced by acid
stress and glutamate, arginine and lysine, respectively. Their endproducts, gamma aminobutyric acid
(GABA), agmatine or cadaverine, are exported out of the cell, thus increasing the external pH. In
addition, regulators such as Fur, PhoP and RpoS induce the expression of specific acid shock proteins
3 Additionally, Acid Resistance systems (AR) contribute to the survival of E. coli at a pH of 2 for at least two hours (Foster, 2004; Gorden & Small, 1993). There are five different systems known to help bacteria respond to many different acid stress conditions. In the glucose repressed AR1 system, which is also known as the oxidative system (Bearson et al., 1997), the sigma factor σS encoded by the gene rpoS and the cAMP receptor protein CRP regulate the expression of several ASPs. The mechanism is still unclear, but AR1 helps cells which were grown to stationary phase in pH 5.5 buffered Lysogeny Broth (LB) medium survive the
shift to pH 2.5 buffered minimal medium (Foster, 2004). The systems AR2, AR3 and AR4, also called the Gad (AR2), Adi (AR3) or Cad (AR4) system, require all the activity of an enzyme to decarboxylate the amino acids (aa) glutamate, arginine or lysine, respectively. Within this enzymatic reaction catalyzed by the decarboxylases GadA/GadB, AdiA or CadA, CO2 is produced and a cellular proton is consumed, thus contributing to an increase of the internal pH. The resulting basic polyamines gamma aminobutyric acid (GABA), agmatine or cadaverine are exported out of the cell by the amino acid/polyamine antiporters GadC, AdiC or CadB, leading to an increase of the extracellular pH (Foster, 2004). AR2, the best known of the AR systems, and AR3 can be partially induced by σS and enable cell survival at stationary phase and pH values as low as 2.5 by maintaining an internal pH of 4.2 and 4.7, respectively (Richard & Foster, 2004). By contrast, the pH optimum of the AR4 lysine decarboxylase is higher and lies at pH 5.7 (Sabo et al., 1974). Furthermore, AR3 is only active under anaerobic conditions, while AR4 favors microaerobic conditions. In some E. coli strains, but not in K12 strains, the AR5 system exists. This system contains SpeF, an ornithine decarboxylase, and PotE, an ornithine-putrescine antiporter. Like AR4, it functions only under moderately acidic conditions (Applebaum et al., 1977; Zhao & Houry, 2010).
As periplasmic proteins also need acid stress protection, E. coli expresses acid inducible chaperones such as HdeA and HdeB, which act in the periplasm by protecting proteins from
4
1.2.
The Cad system
As mentioned above, E. coli possesses the lysine dependent decarboxylase system AR4: the Cad system (Fig. 2). Its main components are the lysine decarboxylase CadA, the lysine/cadaverine antiporter CadB and the regulator of the cadBA operon – CadC (Auger et al., 1989; Meng & Bennett, 1992a; Meng & Bennett, 1992b; Sabo et al., 1974; Watson et al., 1992).
1.2.1. CadA, CadB and CadC
In E. coli, two different types of amino acid decarboxylases exist: Constitutively expressed biosynthetic decarboxylases important for polyamine synthesis (Goldemberg, 1980; Tabor & Tabor, 1985) and inducible biodegradative decarboxylases like CadA (Gale, 1946). The activity of the cytoplasmic CadA reaches its maximal level when incubated in rich medium containing lysine with low oxygen levels and acidic pH (Sabo et al., 1974). The enzyme catalyzes the decarboxylation of lysine to cadaverine while consuming a proton and releasing CO2. It has a relative size of 81 kDa (715 aa) and forms decamers consisting of five dimers (Kanjee et al., 2011; Meng & Bennett, 1992a; Sabo et al., 1974). Two CadA decamers and up to five hexamers of the AAA+-ATPase RavA (ATPases associated with various cellular activities) can build a cage-like complex, which does not influence CadA activity, but stimulates the ATPase activity of RavA (Snider et al., 2006). Furthermore, an interaction with the small alarmone ppGpp blocks CadA activity (Kanjee et al., 2011). Since ppGpp responds to amino acid starvation in the cell, it presumably inhibits the lysine decarboxylase to prevent further lysine consumption.
Following lysine decarboxylation, the end product cadaverine is exported by the
lysine/cadaverine antiporter CadB (Meng & Bennett, 1992b). CadB is an inner membrane protein with a relative size of 47 kDa (444 aa) and contains 12 transmembrane helices (Meng
& Bennett, 1992a). Furthermore, it shows similarity to the ornithine/putrescine antiporter PotE (Soksawatmaekhin et al., 2004). The exported polyamine cadaverine not only contributes to the increase of the extracellular pH, but also inhibits the activity of the porines OmpC and OmpF, thus decreasing of proton influx (Samartzidou & Delcour, 1999; Samartzidou et al., 2003).
The genes cadC, cadB and cadA are clustered on the E. coli chromosome, while cadB and
5 expression under low pH conditions and in the presence of lysine (Neely et al., 1994). CadC is anchored in the inner membrane and has a relative molecular mass of 58 kDa (512 aa) (Dell
et al., 1994; Watson et al., 1992). It consists of three different domains: the cytoplasmic N-terminal domain (aa 1 - 158), the transmembrane domain (aa 159 - 187) and the periplasmic C-terminal domain (aa 188 - 512) (Dell et al., 1994). CadC belongs to the ToxR-like
transcriptional activators, as approximately 100 amino acids of the NH2-terminus show high similarity to the COOH-termini of other ToxR-like regulators such as ToxR and Tcp of Vibrio
cholera, PsaE of Yersinia pseudotuberculosis and WmpR of Pseudoalteromonas tunicata
(Egan et al., 2002; Hase & Mekalanos, 1998; Miller et al., 1987; Watson et al., 1992; Yang & Isberg, 1997). Like ToxR, CadC is not part of a two-component-system consisting of a kinase and a response regulator, but rather acts as one-component system with DNA-binding at its NH2-terminus (Miller et al., 1987; Ulrich et al., 2005; Watson et al., 1992).
1.2.2. Regulation of the Cad system
Several different mechanisms exist to regulate the expression of the cad genes cadA and cadB
(Fig. 2). Mutations in the periplasmic domain of CadC revealed that CadC acts as the pH sensor of the system (Dell et al., 1994; Haneburger et al., 2011). In addition, two periplasmic cysteine residues (Cys208 and Cys272) can build a disulfide bridge at neutral pH, which is reduced at low pH. Therefore, cysteines seem to play a functional role in the pH sensing of CadC, too (Tetsch et al., 2011). Detection of the two CadC binding sites Cad1 (-144 to -112 bp) and Cad2 (-89 to -59 bp) in the cadBA promoter (Pcad or PcadBA), crosslinking-studies and
the crystallization of the periplasmic domain led to speculations that CadC binds to the promoter as an oligomer (Eichinger et al., 2011; Küper & Jung, 2005). Furthermore, PcadBA
harbors five binding sites for the small histone-like protein H-NS (Küper & Jung, 2005), and also the promoter of CadC is regulated by H-NS (Krin et al., 2010). H-NS binding causes altered DNA topology and transcriptional repression at neutral pH and aerobic conditions
(Cortassa & Aon, 1993; Reams et al., 1997; Shi et al., 1993). Anaerobiosis leads to ten times higher CadA activities as aerobiosis (Sabo et al., 1974). In the absence of lysine, the Cad
6 exogenous lysine, lysine is transported into the cell by LysP leading to reduced interaction with CadC and to cadBA expression at acidic pH (Tetsch et al., 2008).
Figure 2: The Cad system of E. coli. At neutral pH and in the absence of lysine (in red), the lysine
permease LysP inhibits the regulator CadC. Additionally, H-NS prevents cadBA expression. A
decrease of the external pH, together with the presence of lysine, leads to a release of CadC and lysine
transport into the cell by LysP. CadC can bind to the PcadBA promoter, thereby starting the production
of the lysine decarboxylase CadA and the antiporter CadB. CadA decarboxylases lysine while
consuming a proton and producing CO2 and cadaverine.
7 2008). In addition to these findings, the deletion of the gene yjeK resulted in a CadA negative phenotype (Kraxenberger, 2006). YjeK in concert with YjeA has been reported to post-translationally modify the translation elongation factor EF-P (Bailly & de Crecy-Lagard, 2010).
1.3.
Steps in translation
Ribosomes translate mRNAs into the corresponding amino acid sequences of proteins (Liljas, 2009). They consist of several rRNAs and ribosomal proteins and make up the 70S complex with the two subunits 30S and 50S in bacteria. In the process of protein synthesis, t-RNA synthetases are needed to ligate amino acids to the 3´CCA-end of their accordant transfer RNAs (tRNAs) (Schimmel, 1987). After the transport of the resulting aminoacyl-tRNAs to the ribosome, a plethora of initiation, elongation and termination factors are recruited to run the protein synthesis. To start translation, the 30S ribosome interacts with the mRNA at its Shine Dalgarno sequence upstream of the start codon AUG [reviewed in (Schmeing & Ramakrishnan, 2009)]. The initiation factors IF-1, IF-2 and IF-3 help the initiator tRNA N-formyl-Met-tRNA (fMet-tRNAi) to localize at the P (peptidyl)-site of the 30S ribosome under GTP consumption by combining the anticodon binding site of the tRNA with the AUG codon on the mRNA (Fig. 3A). Subsequently, the initiation factors are released and the stable 70S complex is built. With the help of elongation factor EF-Tu and GTP consumption, the second amino acid is placed at the A (aminoacyl)-site of the ribosome followed by the formation of the first peptide bond. This step is catalyzed by the peptidyl transferase center of the ribosomal 50S subunit (Fig. 3B). The elongation factor EF-G is then needed to translocate the fMet-tRNAi to the ribosomal E (exiting)-site and the second amino acid to the ribosomal
P-site followed by a release of the fMet-tRNA (Fig. 3C, D). Finally, the termination factors RF-1, RF-2 and RF-3 are important to recognize the stop codon on the mRNA, to release the
8 Figure 3: Translation initiation and elongation [reviewed in (Schmeing & Ramakrishnan, 2009)].
A) The initiation factors IF-1, IF-2 and IF-3 help the initiator tRNA N-formyl-Met-tRNA
(orange) bind with its anticodon binding site to the start codon AUG, which is localized at the P
(peptidyl)-site of the 30S ribosome. After the release of the initiation factors, the 70S complex is
formed.
B) The second aminoacyl-tRNA (purple) is placed to the A (aminoacyl)-site of the ribosome
by the help of the elongation factor EF-TU. In a peptidyltransferase reaction, the amino acids are
linked.
C) The elongation factor EF-G helps the deacetylated initiator tRNA (orange) to translocate to
the E (exit)-site. The second aminoacyl-tRNA (purple) moves to the P-site. The third aminoacyl-tRNA
is shown in yellow.
D) The initiator tRNA (orange) is released from the E-site, and the next aminoacyl-tRNA
(yellow) can occupy the A-site. The amino acids are linked by the peptidyltransferase activity of the
9
1.4.
The elongation factor EF-P
EF-P has been extensively investigated for more than 30 years (Glick & Ganoza, 1975). The protein has a size of 20.5 kDa (187 aa) and is highly conserved in all bacteria (Aoki et al., 1991; Kyrpides & Woese, 1998). As EF-P was suggested to be essential for cell viability, mainly in vitro studies have been performed (Aoki et al., 1997a; Aoki et al., 1997b; Ganoza & Aoki, 2000; Glick et al., 1979). However, subsequent studies described the generation and analysis of efp mutants in various bacterial strains (Baba et al., 2006; Kearns et al., 2004; Peng et al., 2001; Zou et al., 2011). The characterization of these mutants revealed the importance of EF-P for swarming and sporulation in Bacillus subtilis (Kearns et al., 2004) as well as for virulence and stress response in Agrobacterium tumefaciens and Salmonella enterica (Peng et al., 2001; Zou et al., 2011). Although EF-P is known to have a stimulatory
effect on peptide bond formation between the ribosome-bound initiator transfer RNA fMet-tRNAi and the tRNA analogue puromycin (Aoki et al., 1991; Glick & Ganoza, 1975), the protein is not an essential component of in vitro translation systems (Shimizu et al., 2001). In
E. coli, one EF-P copy per ten ribosomes can be found, which resembles the copy number of other initiation factors (An et al., 1980; Cole et al., 1987). However, the detection of EF-P in monosome as well as in polysome fractions led to suggestions that EF-P not only promotes formation of the first peptide bond, but also plays a role during translation elongation (Aoki et al., 2008).
Archaea and eukaryotes possess the orthologous variants aIF-5A and eIF-5A, which share 84% and 64% sequence identity, respectively (Bartig et al., 1992; Cooper et al., 1983). In
mammalian cells, the genes encoding eIF-5A1 and its isoform eIF-5A2 are oncogenes (Clement et al., 2003). Furthermore, eIF5a is suggested to play a role in HIV protein Rev mediated transport of mRNAs out of the nucleus, mRNA decay and cell cycle progress (Bevec et al., 1996; Park et al., 1997; Ruhl et al., 1993; Zuk & Jacobson, 1998) as well as in diabetes and malaria (Kaiser, 2012).
10 alternative modification has been reported for EF-P (Aoki et al., 2008; Bailly & de Crecy-Lagard, 2010; Navarre et al., 2010; Peil et al., 2012) (Fig. 4).
Figure 4: The EF-P modification pathway. YjeK converts (S)-α-lysine into (R)-β-lysine, which is then transferred to the K34 of E. coli EF-P by YjeA and hydroxylated by YfcM. Lysylated EF-P gains
an additional mass of 128 Da, hydroxylated EF-P of 144 Da.
The two genes yjeA and yjeK are often organized in one cluster in bacterial genomes, which has led to the finding that YjeK and YjeA are involved in EF-P modification (Bailly & de Crecy-Lagard, 2010; Yanagisawa et al., 2010). In a first step, the iron sulfur protein YjeK acts as a 2,3-lysine aminomutase (LAM) while catalyzing the isomerisation of (S)-α-lysine to
(R)-β-lysine (Behshad et al., 2006). The (R)-β-lysine is then activated and transferred to the conserved lysine residue 34 (K34) of EF-P by YjeA resulting in a modified and thus active EF-P (Gilreath et al., 2011; Roy et al., 2011). YjeA (also known as PoxA or GenX) is a homolog to the class II lysyl-tRNA-synthetase LysRS. In E. coli, YjeA shows 32% identity and 50% homology to the COOH-terminus of LysRS containing the catalytic core important for lysine binding, but it lacks the NH2-terminus of LysRS with the anticodon binding site important for tRNA binding (Ambrogelly et al., 2010; Navarre et al., 2010). Therefore, it has been suggested that YjeA has a function differing from the aminoacylation of tRNAs (Bailly & de Crecy-Lagard, 2010). Drawing on mass spectrometry (MS) analysis for modified EF-P (Aoki et al., 2008), Peil et al. have reported recently that K34 of EF-P is additionally hydroxylated by the O2 dependent hydroxylase YfcM (Peil et al., 2012) (Fig. 4). However, a
deletion of yfcM leads to a wildtype like phenotype in Salmonella enterica, which indicates that the hydroxylation plays a minor role in vivo (Bullwinkle et al., 2013).
11 to the P-site of the ribosome (P-tRNAs) and stimulates peptide bond formation through stabilization and positioning of the acceptor end of the P-tRNAs (Blaha et al., 2009). In addition, several further functions of EF-P have been suggested, including the stimulation of the first peptide bond (Glick et al., 1979), the coupling of the ribosomal 30S and 50S subunits (Aoki et al., 1991), the enhancement of polylysine synthesis (Glick & Ganoza, 1976), the
change of the conformation of the ribosomal active site (Ganoza & Aoki, 2000) and the inhibition of tRNAs to bind to the A-site before ribosomal proofreading (Aoki et al., 2008).
As the overall protein synthesis is diminished by only 30% in yeast cells, it has been assumed that eIF5A does not play a general role in the translational machinery, but rather has importance only for a small subset of mRNAs (Kang & Hershey, 1994). For example, the translation of the porin KdgM is dependent on EF-P in Salmonella, thus a deletion of efp
results in increased membrane permeability and in various phenotypes like attenuated virulence in the bacterium (Zou et al., 2011). Although plenty suggestions have been made for the action mode of EF-P, in vivo data are still missing.
1.5.
Scope of the thesis
In this study, the relevance of elongation factor P and its modifying proteins YjeK and YjeA in regard to the Cad system is analyzed in more detail. To this end, yjeA/yjeK and efp mutants are characterized by determining their lysine decarboxylase phenotype, their capability for
cadBA expression under non- and inducing conditions and their ability to interact with each other and other components of the Cad system.
Additionally it is examined, which of the Cad proteins is dependent on EF-P mediated translation. As no direct EF-P - target has ever been identified before in E. coli, the finding of
the first target can greatly improve the understanding of the function of EF-P.
Furthermore it is investigated, if EF-P recognizes a special signal sequence. With the signal
sequence in hand, further targets can be identified. As the physiological role of EF-P is still unclear, the signal sequence can provide first insights into the mode of action of EF-P in vivo. To confirm the importance of EF-P for the translation of target proteins, in vitro translation assays are performed by Dr. Daniel Wilson and Dr. Agata Starosta (Gene Center, LMU Munich).
12
2.
Materials and Methods
2.1.
Materials
Following materials were used in this study:
Table 1: Materials and their manufacturers used in this study.
Material Manufacturer
[γ-32P] ATP Perkin Elmer (Rodgau)
5-Bromo-4-Chloro-3-Indoxyl Phosphate (BCIP) AppliChem (Darmstadt)
Acrylamide Protogel Biozym Diagnostik (Hessisch Oldendorf)
Agarose Serva (Heidelberg)
Alkaline Phosphatase New England Biolabs (Frankfurt)
Conjugated anti-Mouse IgG GE Healthcare (Braunschweig)
Ammonium Persulfate (APS) National Diagnostics (Atlanta)
Ampicillin (sodium salt) Roth (Karlsruhe)
Anti-Rabbit IgG GE Healthcare(Braunschweig)
Calf Intestinal Phosphatase (CIP) New England Biolabs (Frankfurt)
Carbenicillin (disodium salt) Roth (Karlsruhe)
Chloramphenicol Sigma (Deisenhofen)
Chloroform Roth (Karlsruhe)
Diethylpyrocarbonate (DEPC) Roth (Karlsruhe)
DNA oligonucleotides Sigma (Deisenhofen)
Invitrogen (Karlsruhe)
Eurofins MWG Operon (Ebersberg)
DNase I Sigma (Deisenhofen)
DNA standard (2-Log DNA-Ladder) New England Biolabs (Frankfurt)
DNeasy 96 blood and tissue kit Qiagen (Hilden)
dNTPs (deoxynucleotide triphosphates) Invitrogen (Karlsruhe)
Glycerol Roth (Karlsruhe)
13
Hi Yield Plasmid Mini Kit Süd-Laborbedarf (Gauting)
Hybond-P protein transfer membrane GE Healthcare (Braunschweig)
Isopropyl-D-thiogalactopyranoside (IPTG) PeqLab (Erlangen)
Kanamycin (sulfate) Roth (Karlsruhe)
L-(+) - Arabinose Roth (Karlsruhe)
L-Lysine Sigma (Deisenhofen)
Nitro Blue Tetrazolium (NBT) Biomol (Hamburg)
Ni-NTA agarose Qiagen (Hilden)
Ni-NTA magnetic agarose beads Qiagen (Hilden)
Nitrocellulose membrane GE Healthcare (Braunschweig)
Page Ruler Prestained Protein Ladder Plus Fermentas (St. Leon-Rot)
Penta-His anti-Mouse-IgG Qiagen (Hilden)
Phenol-Chloroform Roth (Karlsruhe)
Phenol/Chloroform/Isoamylalcohol (25/24/1) Roth (Karlsruhe)
Phenylmethylsulfonyl fluoride (PMSF) Sigma (Deisenhofen)
Phusion DNA Polymerase Finnzyme (Espoo, Finnland)
Pyridoxal Phosphate (PLP) Fluka (Neu-Ulm)
Quick & Easy E. coli Gene Deletion Kit Gene Bridges (Heidelberg)
Restriction Nuclease New England Biolabs (Frankfurt)
Bovine Serum Albumin (BSA) AppliChem (Darmstadt)
Spermidine Fluka (Neu-Ulm)
T4 DNA Ligase New England Biolabs (Frankfurt)
Taq-DNA Polymerase Peqlab (Erlangen)
Thermoscript Reverse Transcriptase Invitrogen (Karlsruhe)
Trinitrobenzenesulfonic acid (TNBS) Sigma (Deisenhofen)
Triton X-100 Calbiochem (La Jolla, Kalifornien)
Tween 20 GE Healthcare (Braunschweig)
14
2.2.
Strains, plasmids and oligonucleotides
Strains used in this study are listed in Table 2, plasmids in Table 3 and oligonucleotides in Table 4.
Table 2: Strains used in this study. All strains are Escherichia coli derivates.
Strain Feature Source
DH5α
F– Φ80lacZ∆M15 ∆(lacZYA-argF) U169 recA1 endA1 hsdR17
(rK–, mK+) phoA supE44 λ–thi-1 gyrA96 relA1 Promega
MG1655 E. coli K-12 reference strain (Blattner et al., 1997)
P1#36 MG1655 yjeK643-1029::Tn10 (Kraxenberger, 2006)
TK1 MG1655 yjeK643-1029::npt (Kraxenberger, 2006)
BW25113
∆(araD-araB)567, ∆lacZ4787(::rrnB-3), lambda-, rph-1, ∆
(rhaD-rhaB)568, hsdR514 (Baba et al., 2006)
JW4116 BW25113 yjeA::npt (Baba et al., 2006)
JW4106 BW25113 efp::npt (Baba et al., 2006)
JW4094 BW25113 cadC::npt (Baba et al., 2006)
MG-CL-1 MG1655 ∆lacZ::tetrpsL150 ∆cadAcadA::lacZ this study
MG-CR MG1655 ∆lacZ::tetrpsL150 ∆cadBAcadBA::lacZ (Ruiz et al., 2011)
MG-CL 1-3 MG1655 ∆lacZ::tetrpsL150 cadC::npt ∆cadCBAcadBA::lacZ (Schüppel, 2010)
MG-CL-12-yjeA MG1655 ∆lacZ::tetrpsL150 yjeA::npt ∆cadBAcadBA::lacZ this study
MG-CL-12-yjeK MG1655 ∆lacZ::tetrpsL150 yjeK642-1029::npt ∆cadBAcadBA::lacZ this study
MG-CL-12-hflx MG1655 ∆lacZ::tetrpsL150 hflx1-750::npt ∆cadBAcadBA::lacZ this study
MG-CL-12-hfq MG1655 ∆lacZ::tethfq1-230::cat ∆cadBAcadBA::lacZ this study
SU1 BW25113 yjeK643-1029::cat this study
BL21 (DE3) E. coli BF-dcm ompT hsdS (rB-mB-) gal
(Studier & Moffatt, 1986)
SU2 MG1655 yjeA::npt this study
SU3 BL21 (DE3) yjeK643-1029::cat this study
SU4 JW4106 hns::cat this study
15 Table 3: Plasmids used in this study. Plasmid constructions are further explained in the middle
column.
Plasmid Feature / Construction comments Source
General vectors
pET16b Ampr-cassette, T7-promoter, pBBR322 origin, 5‘-His-tag coding sequence, lacI-coding sequence, lac operator
Novagen (Merck Millipore) pBAD24 Ampr-cassette, pBBR322 origin, araC coding sequence, ara operator (Guzman et al.,
1995) pBBR1-MCS5 Gmr-cassette, pBBR broad host range origin of replication, mob region
for conjugative transfer
(Kovach et al., 1995)
pBBR1-MCS3-LacZ
Tetr-cassette, pBBR broad host range origin of replication, mob region for conjugative transfer, lacZ coding sequence
(Fried et al., 2012) pQE70 Ampr-cassette, 5‘-His-Tag coding sequence, lacI-coding sequence, lac
operator
Qiagen
pIVEX2.3MCS Ampr-cassette, T7-promoter, 3‘-His-Tag coding sequence Roche pQE70-Eco_efp efp cloned using SphI, BamHI restrictions sites (Peil et al.,
2012) pQE70-Eco_efp
K34A/
pQE70 plasmid harboring efp with a single mutation (K34A) Dr. Agata Starosta pQE70-Eco_efp
K34R
pQE70 plasmid harboring efp with a single mutation (K34R) Dr. Agata Starosta pAF/kamA Ampr-cassette,T7 promoter, pET23a origin (Ruzicka et al.,
2000)
pAlter-Ex2/argU Tetr-cassette, tac promoter, p15a origin (Ruzicka et al., 2000)
pET16b-His6-YjeK
yjeK in pET16b, 5´-His6-tag (Kraxenberger, 2006)
pBAD24-yjeA yjeA in pBAD24, with NdeI and XbaI, 5´-His6-tag, P121+P122 this study pBAD24-yjeK pBAD24 with NcoI-XmaI fragment from pUC19-rbs-his10-yjeK,
5´-His-tag
(Kraxenberger, 2006)
pBAD24-hfq hfq in pBAD24, with NdeI and EcoRI, P119+P120, 3´-His-tag this study
cadC-lacZ promoter fusions
p3LC-TF Fusion of cadC promoter with artificial RBS to lacZ / P1+P3 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PstI)
this study
p3LC-TL02 Fusion of cadC promoter to sequence encoding 2 amino acids of cadC
and lacZ/ P1+P4 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL02 (Ala, Gly, His, Leu, Met, Phe, Pro)
cadC promoter fusion with artificial RBS to lacZ+variabel second aa (Ala, Gly, His, Leu, Met, Phe or Pro)/P1+P111-P117 PCR fragments in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30 Translational CadC’-LacZ fusion (sequence encodes 30 amino acids of
cadC)/ P1+P5 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL66 Translational CadC’-LacZ (sequence encodes 66 amino acids of cadC)/ P1+P6 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL100 Translational CadC’-LacZ fusion (sequence encodes 100 amino acids of
cadC)/ P1+P7 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL108 Translational CadC’-LacZ fusion (sequence encodes 108 amino acids of
cadC)/ P1+P8 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL131 Translational CadC’-LacZ fusion (sequence encodes 131 amino acids of
cadC)/ P1+P9 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL158 Translational CadC’-LacZ fusion (sequence encodes 158 amino acids of
cadC)/ P1+P10 PCR fragment in pBBR1-MCS3-LacZ (XbaI+PciI/NcoI)
this study
p3LC -TL158-CSPPP
Translational CadC´-LacZ fusion; (sequence encodes 158 amino acids of
cadC),S118C substitution in cadC, P11+12 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
16
Plasmid Feature / Construction comments Source
p3LC -TL158-GSPPP
Translational CadC´-LacZ fusion; (sequence encodes 158 amino acids of
cadC), S118G substitution in cadC, P13+P14 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC -TL30-GPP Translational CadC´-LacZ fusion; sequence encodes 30 amino acids of
cadC+sequence encoding GPP, P1+P15 fragmentin pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC -TL30-PGP Translational CadC´-LacZ fusion; (sequence encodes 30 amino acids of
cadC+sequence encoding PGP, P1+P16 fragmentin pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC -TL30-PPG Translational CadC´-LacZ fusion; (sequence encodes 30 amino acids of
cadC+sequence encoding PPG, P1+P17 fragmentin pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
Artificial introduction of three consecutive prolines
p3LC-TL02-P p3LC-TL02 + sequence encoding 1 proline / P1+P18 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL02-3P p3LC-TL02+ sequence encoding 3 prolines / P1+P19 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-3P p3LC-TL30 + sequence encoding 3 prolines / P1+P20 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL66-3P p3LC-TL66 + sequence encoding 3 prolines / P1+P21 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL100-3P p3LC-TL100 + sequence encoding 3 prolines / P1+P22 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL108-3P p3LC-TL108 + sequence encoding 3 prolines / P1+P23 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
Artificial modification of the proline stretch length in CadC´-LacZ TL30
p3LC-TL30-5P p3LC-TL30 + sequence encoding 5 prolines / P1+P24 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-4P p3LC-TL30 + sequence encoding 4 prolines / P1+P25 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-2P p3LC-TL30 + sequence encoding 2 prolines / P1+P26 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-1P p3LC-TL30 + sequence encoding 1 proline / P1+P27 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
Artificial modification of the proline stretch codon usage
p3LC-TL30-3P-CCA
p3LC-TL30 + sequence for 3 x CCA / P1+P28 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-3P-CCG
p3LC-TL30 + sequence for 3 x CCG / P1+P29 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-3P-CCT
p3LC-TL30 + sequence for 3 x CCT / P1+P30 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
Artificial introduction of sequences encoding stretches of various amino acids
p3LC-TL30-5F p3LC-TL30 + sequence encoding 5 phenylalanines / P1+P31 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5G p3LC-TL30 + sequence encoding 5 glycines / P1+P32 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5A p3LC-TL30 + sequence encoding 5 alanines / P1+P33 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5V p3LC-TL30 + sequence encoding 5 valines / P1+P34 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5L p3LC-TL30 + sequence encoding 5 leucines / P1+P35 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5I p3LC-TL30 + sequence encoding 5 isoleucines / P1+P36 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
17
Plasmid Feature / Construction comments Source
p3LC-TL30-5M p3LC-TL30 + sequence encoding 5 methionines / P1+P37 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5S p3LC-TL30 + sequence encoding 5 serines / P1+P38 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5T p3LC-TL30 + sequence encoding 5 threonines / P1+P39 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5D p3LC-TL30 + sequence encoding 5 aspartates / P1+P40 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5E p3LC-TL30 + sequence encoding 5 glutamates / P1+P41 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5Y p3LC-TL30 + sequence encoding 5 tyrosines / P1+P42 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5N p3LC-TL30 + sequence encoding 5 asparagines / P1+P43 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5K p3LC-TL30 + sequence encoding 5 lysines / P1+P44 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5H p3LC-TL30 + sequence encoding 5 histidines / P1+P45 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5Q p3LC-TL30 + sequence encoding 5 glutamines / P1+P46 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5C p3LC-TL30 + sequence encoding 5 cysteines / P1+P47 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5W p3LC-TL30 + sequence encoding 5 tryptophans / P1+P48 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-TL30-5R p3LC-TL30 + sequence encoding 5 arginines / P+P49 PCR fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
Generation of CadC Pro to Ala substitution mutants
p5C cadC native copy including the native promoter / P1+P2 PCR fragment
in pBBR1-MCS5 (XbaI +PspOMI)
this study
p5C-P120A cadC mutant derivative P120A including the native promoter / P50+P51 PCR-OL-fragment in pBBR1-MCS5 (XbaI +PspOMI)
this study
p5C-P121A cadC mutant derivative P121A including the native promoter / P52+P53 PCR-OL-fragment in pBBR1-MCS5 (XbaI +PspOMI)
this study
p5C-P122A cadC mutant derivative P122A including the native promoter / P54+P55 PCR-OL-fragment in pBBR1-MCS5 (XbaI +PspOMI)
this study
p5C-P/A cadC mutant derivative PPPIP/AAAIS including the native promoter / P56+P57 PCR-OL-fragment in pBBR1-MCS5 (XbaI +PspOMI)
this study
p3LC -TL158-P120A
p3LC-TL158 mutant derivative P121A / P1+P10 PCR fragment from p5C-P120A template in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC -TL158-P121A
p3LC-TL158 mutant derivative P121A / P1+P10 PCR fragment from p5C-P121A template in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC -TL158-P122A
p3LC-TL158 mutant derivative P121A / P1+P10 PCR fragment from p5C-P122A template in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC -TL158-P/A
p3LC-TL158 mutant derivative PPPIP/AAAIS / P1+P10 PCR fragment from p5C-P/A template in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
pET16b-CadC cadC native copy under control of IPTG inducible T7 polymerase dependent promote, His6-CadC
(Küper & Jung, 2005)
pET16b-CadC-P/A
cadC mutant derivative PPPIP/AAAIS T7 polymerase dependent / PCR
fragment from p5C-P/A template in pET16b
this study
Translational LacZ fusions of other EF-P target proteins fused to the CadC native promoter
p3LC-cadA Translational CadA-LacZ fusion including complete cadA but replacing
the cadAB promoter by the cadC promoter / 61+62 PCR-OL-fragment in
pBBR1-MCS3-LacZ (XbaI +BspHI/NcoI)
this study
p3LC-cadB Translational CadB-LacZ fusion including the complete cadB but replacing the cadAB promoter by the cadC promoter / 58+59 PCR-OL-Fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
18
Plasmid Feature / Construction comments Source
p3LC-flk Translational FlK-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-amiB Translational AmiB-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-flhC Translational FlhC-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-nlpD Translational NlpD-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-rzoR Translational RzoR-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-tonB Translational TonB-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-uvrB Translational UvrB-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-hisD Translational HisD-LacZ fusion; for construction see table 6 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-flhC-APP Translational FlhC-LacZ fusion; P/A substitute, P83-P84 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-flhC-PAP Translational FlhC-LacZ fusion; P/A substitute, P85+P86 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
p3LC-flhC-AAA Translational FlhC-LacZ fusion; P/A substitute, P87+P88 PCR-OL-fragment in pBBR1-MCS3-LacZ (XbaI +PciI/NcoI)
this study
Bacterial two hybrid studies
pKT25 ori-p15a T25 fragment for C-terminal fusion; Kmr Euromedex pKNT25 ori-p15a T25 fragment for N-terminal fusion; Kmr Euromedex pUT18 ori-ColE1 T18 fragment for N-terminal fusion; Ampr Euromedex pUT18C ori-ColE1 T18 fragment for N-terminal fusion; Ampr Euromedex pUT18-zip Positive control plasmid; encoding for yeast leucine zipper GCN4 Euromedex pKT25-zip Positive control plasmid; encoding for yeast leucine zipper GCN4 Euromedex pKT25-CadC cadC in pKT25 (XbaI+BamHI), P166+P167 (Krönauer,
2011) pKT25-LysP lysP in pKT25, (XbaI+BamHI), P168+P169 (Krönauer,
2011) pKT25-YjeA yjeA in pKT25, (XbaI+BamHI), P170+P171 (Krönauer,
2011) pKT25-YjeK yjeK in pKT25, (XbaI+BamHI), P174+P175 (Krönauer,
2011) pKT25-EFP efp in pKT25, (XbaI+BamHI), P164+P165 (Krönauer,
2011) pKT25-HNS hns in pKT25, (XbaI+BamHI), P172+P173 (Krönauer,
2011) pKTN25-CadC cadC in pKTN25, (XbaI+BamHI), P166+P167 (Krönauer,
2011) pKTN25-LysP lysP in pKTN25, (XbaI+BamHI), P168+P169 (Krönauer,
2011) pKTN25-YjeA yjeA in pKTN25, (XbaI+BamHI), P170+P171 (Krönauer,
2011) pKTN25-YjeK yjeK in pKTN25, (XbaI+BamHI), P174+P175 (Krönauer,
2011) pKTN25-EFP efp in pKTN25, (XbaI+BamHI), P164+P165 (Krönauer,
2011) pKTN25-HNS hns in pKTN25, (XbaI+BamHI), P172+P173 (Krönauer,
2011) pUT18C-CadC cadC in pUT18C, (XbaI+BamHI), P166+P167 (Krönauer,
2011) pUT18C-LysP lysP in pUT18C, (XbaI+BamHI), P168+P169 (Krönauer,
19
Plasmid Feature / Construction comments Source
pUT18C-YjeA yjeA in pUT18C, (XbaI+BamHI), P170+P171 (Krönauer, 2011) pUT18C-YjeK yjeK in pUT18C, (XbaI+BamHI), P174+P175 (Krönauer,
2011) pUT18C-EFP efp in pUT18C, (XbaI+BamHI), P164+P165 (Krönauer,
2011) pUT18C-HNS hns in pUT18C, (XbaI+BamHI), P172+P173 (Krönauer,
2011)
Generation of CadC Ly to Ala mutants
pET16b-K59A cadC mutant derivate K59A under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K77A cadC mutant derivate K77A under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K95A cadC mutant derivate K95A under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K99A cadC mutant derivate K99A under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K80A cadC mutant derivate K80A under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
Generation of CadC Ly to Arg mutants
pET16b-K59R cadC mutant derivate K59R under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K77R cadC mutant derivate K77R under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K95R cadC mutant derivate K95R under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K99R cadC mutant derivate K99R under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
pET16b-K80R cadC mutant derivate K80R under control of IPTG inducible T7 polymerase dependent promoter/PCR-OL-fragment in pET16b-CadC
this study
PcadA-lacZ fusions
P3C-PrA79 pBBR1-MCS3-LacZ harboring 79 bp of the sequence upstream of cadA
+1, P200+P209
this study
P3C-PrA100 pBBR1-MCS3-LacZ harboring 100 bp of the sequence upstream of
cadA +1, P201+P209
this study
P3C-PrA200 pBBR1-MCS3-LacZ harboring 200 bp of the sequence upstream of
cadA +1, P202+P209
this study
P3C-PrA300 pBBR1-MCS3-LacZ harboring 300 bp of the sequence upstream of
cadA +1, P203+P209
this study
P3C-PrA400 pBBR1-MCS3-LacZ harboring 400 bp of the sequence upstream of
cadA +1, P204+P209
this study
P3C-PrA500 pBBR1-MCS3-LacZ harboring 500 bp of the sequence upstream of
cadA +1, P205+P209
this study
P3C-PrA600 pBBR1-MCS3-LacZ harboring 600 bp of the sequence upstream of
cadA +1, P206+P209
this study
P3C-PrA700 pBBR1-MCS3-LacZ harboring 700 bp of the sequence upstream of
cadA +1, P207+P209
this study
P3C-PrA800 pBBR1-MCS3-LacZ harboring 800 bp of the sequence upstream of
cadA +1, P208+P209
20 Table 4: Oligonucleotides used in this study. All primers were purchased from Sigma Aldrich and
diluted in 1xTE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) for storage at -20°C and -80°C.
Name Sequence
Restric-tion site Source
cadC-lacZ promoter fusions
P1 XbaI-CadC-NP-Fw AGT CTA GAC CTG AGC TAT AGC
ACT AAC TGA CG XbaI this study
P2 PspOMI-CadC-Rev CCG GGC CCA AAC TCA ACA ACA AAT ATT TCC GAG CAT A PspOMI this study
P3 PstI-CadC-TriF-Rev
GCC TGC AGA ATA GAA ACT CAT
TCG AAA AGG GAA TGA TG PstI this study
P4 PciI-CadC-TF02-Gln-Rev
GCA CAT GTG TTG CAT AAT AGA
AAC TCA TTC GAA AAG GG PciI this study
P5 PciI-TF30E-CadC-Rev GCA TAC ATG TGC TCA AGG GTA
AGT TGA CGC CCA TTG CG PciI this study
P6 PciI-TF66-CadC-Rev GCA CAT GTG ATT GGT GAC AAT
ACT TCT CTT CCA G PciI this study
P7 PciI-TF100-CadC-Rev GCA CAT GTT TAA TTT ATA GCC
GCG CTT TGG TAC A PciI this study
P8 PciI-CadC-LacZ-TL108-Rev
CGA CAT GTG GCT GTA CCA GAT
AAC CGG CAC C PciI this study
P9 PciI-TF131-CadC-Rev GAA CAT GTC TGT GGC AGG AAC CGC CTC PciI this study
P10 PciI-TF158-CadC-Rev GCA CAT GTA GGT AGT GAA TCG TTT GCT TTT AAC TGG PciI this study
P11 CadC-CSPPP-fw GAA ATA ATG CTA TGT TCG CCT CCC this study
P12 CadC-CSPPP-rev GGG AGG CGA ACA TAG CAT TAT TTC this study
P13 CadC-GSPPP-fw GGA AAT AAT GCT AGG TTC GCC TCC this study
P14 CadC-GSPPP-rev GGA GGC GAA CCT AGC ATT ATT TCC this study
P15 PciI-TL30-CadC-GPP-Rev
GTA CAT GTG CGG CGG GCC CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P16 PciI-TL30-CadC-PGP-Rev
GTA CAT GTG CGG GCC CGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P17 PciI-TL30-CadC-PPG-Rev
GTA CAT GTG GCC CGG CGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
Artificial introduction of three consecutive prolines
P18 PciI-CadC-TF02-Pro-Rev
GCA CAT GTG CGG CAT AAT AGA
AAC TCA TTC GAA AAG GG PciI this study
P19 PciI-CadC-TF02-3xPro-Rev
GCA CAT GTG TGG TGG AGG CAT AAT AGA AAC TCA TTC GAA AAG GG
PciI this study
P20 PciI-TF30-CadC-3Pro-Rev
GTA CAT GTG TGG TGG AGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P21 PciI-TF66-CadC-3xPro-Rev
GCA CAT GTG TGG TGG AGG ATT
GGT GAC AAT ACT TCT CTT CCA G PciI this study
P22 PciI-TF100-CadC-3xPro-Rev
GCA CAT GTT TGG TGG AGG TAA
TTT ATA GCC GCG CTT TGG TAC A PciI this study
P23 PciI-CadC-LacZ-TL108-3xPro-Rev
GCA CAT GTG TGG TGG AGG GCT
21
Name Sequence
Restric-tion site Source
Artificial modification of the proline stretch length in CadC´-LacZ TL30
P24 PciI-TF30-CadC-5Pro-Rev
GTA CAT GTG TGG TGG AGG TGG TGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P25 PciI-TF30-CadC-4Pro-Rev
GTA CAT GTG TGG TGG AGG TGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P26 PciI-TF30-CadC-2Pro-Rev
GTA CAT GTG TGG TGG CTC AAG
GGT AAG TTG ACG CCC ATT GCG PciI this study
P27 PciI-TF30-CadC-1Pro-Rev
GTA CAT GTG TGG CTC AAG GGT
AAG TTG ACG CCC ATT GCG PciI this study
Artificial modification of the proline stretch codon usage
P28 PciI-TF30-CadC-3Pro-CCA-Rev
GTA CAT GTG TGG TGGTGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P29 PciI-TF30-CadC-3Pro-CCT-Rev
GTA CAT GTG AGG AGGAGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P30 PciI-TF30-CadC-3Pro-CCC-Rev
GTA CAT GTG GGG GGGGGG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
Artificial introduction of stretches of all other amino acids
P31 PciI-TF30-CadC-5Phe-Rev
GTA CAT GTG GAA GAA AAA GAA GAA CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P32 PciI-TF30-CadC-5Gly-Rev
GTA CAT GTG ACC GCC ACC GCC ACC CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P33 PciI-TF030-CadC-5Ala-Rev
GTA CAT GTG CGC GGC CGC GGC CGC CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P34 PciI-TF30-CadC-5Val-Rev
GTA CAT GTG CAC AAC CAC AAC CAC CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P35 PciI-TF30-CadC-5Leu-Rev
GTA CAT GTG CAG CAGCAGCAGCAG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P36 PciI-TF30-CadC-5Ile-Rev
GTA CAT GTG AAT GAT AAT GAT AAT CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P37 PciI-TF30-CadC-5Met-Rev
GTA CAT GTG CAT CATCATCATCAT CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P38 PciI-TF30-CadC-5Ser-Rev
GTA CAT GTG CGA CGA TGA CGA TGA CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P39 PciI-TF30-CadC-5Thr-Rev
GTA CAT GTG GGT CGT GGT CGT GGT CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P40 PciI-TF30-CadC-5Asp-Rev
GTA CAT GTG ATC GTC ATC GTC ATC CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P41 PciI-TF30-CadC-5Glu-Rev
GTA CAT GTG CTC TTC CTC TTC CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P42 PciI-TF30-CadC-5Tyr-Rev
GTA CAT GTG ATA GTA ATA GTA ATA CTC AAG GGT AAG TTG ACG CCC ATT GCG
22
Name Sequence
Restric-tion site Source
P43 PciI-TF30-CadC-5Asn-Rev
GTA CAT GTG ATT GTT ATT GTT ATT CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P44 PciI-TF30-CadC-5Lys-Rev
GTA CAT GTG TTT CTT TTT CTT TTT CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P45 PciI-TF30-CadC-5His-Rev
GTA CAT GTG ATG GTG ATG GTG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P46 PciI-TF30-CadC-5Gln-Rev
GTA CAT GTG CTG TTG CTG TTG CTG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P47 PciI-TF30-CadC-5Cys-Rev
GTA CAT GTG GCA ACA GCA ACA GCA CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P48 PciI-TF30-CadC-5Trp-Rev
GTA CAT GTG CCA CCACCACCACCA CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
P49 PciI-TF30-CadC-5Arg-Rev
GTA CAT GTG GCG ACG GCG ACG GCG CTC AAG GGT AAG TTG ACG CCC ATT GCG
PciI this study
Generation of CadC Pro to Ala substitution mutants
P50 CadC-OL-120A-fw TGC TAT CTT CGG CTC CCC CTA TAC C this study
P51 CadC-OL-120A-rev GGT ATA GGG GGA GCC GAA GAT AGC A this study
P52 CadC-P121A-Whobble-fw
CTA TCT TCG CCT GCC CCT ATA CCA
G this study
P53 CadC-P121A-Whobble-rev
GGT ATA GGG GGA GCC GAA GAT
AGC A this study
P54 CadC-OL-122A-fw TCT TCG CCT CCC GCT ATA CCA
GAG this study
P55 CadC-OL-122A-rev CTC TGG TAT AGC GGG AGG CGA
AGA this study
P56 CadC(Pro/Ala)-OL-fw GCG GCGGCG ATA TCG GAG GCG
GTT CCT GCC ACA G this study
P57 CadC(Pro/Ala)-OL-rev CTG TGG CAG GAA CCG CCT CCG
ATA TCG CCG CCG C this study
Translational LacZ fusions of other EF-P target proteins fused to the CadC native promoter
P58 NP-CadC-cadB-OL-Rev GCA GAA CTC ATA ATA GAA ACT CAT TCG AAA AGG GAA TGA TG this study
P59 NP-CadC-cadB-OL-Fw GAG TTT CTA TTA TGA GTT CTG CCA
AGA AGA TCG G this study
P60 PciI-cadB-TAA-Rev CAC ATG TAA TGT GCG TTA GAC GCG
GTG T PciI this study
P61 NP-CadC-cadA-OL-Rev GCA ATA ACG TTC ATA ATA GAA ACT
CAT TCG AAA AGG GAA TGA TG this study
P62 NP-CadC-cadA-OL-Fw GAG TTT CTA TTA TGA ACG TTA TTG
CAA TAT TGA ATC ACA TG this study
P63 BspHI-cadA-TAA-Rev GCT CAT GAA TTT TTT GCT TTC TTC
TTT CAA TAC CTT AAC BspHI this study
P64 PciI-amiB-rev CAC ATG TCG TTT GGC AGC GTG CGA
TCT GG PciI this study
P65 flhC-OL-Fw GTT TCT ATT ATG AGT GAA AAA AGC ATT GTT CAG GAA GC this study
P66 flhC-OL-Rev CTT TTT TCA CTC ATA ATA GAA ACT CAT TCG AAA AGG GAA TGA T this study
P67 PciI-flhC-rev CAC ATG TCA ACA GCC TGT ACT CTC TGT TCA PciI this study
23
Name Sequence
Restric-tion site Source
P69 nlpD-OL-Rev GAA CAG CCC ATA ATA GAA ACT CAT
TCG AAA AGG GAA T this study
P70 PciI-nlpD-rev CAC ATG TCT CGC TGC GGC AAA TAA
CGC AGC PciI this study
P71 rzoR-OL-Fw GTT TCT ATT ATG TGC ACA TCA AAG
CAG TCT GTC this study
P72 rzoR-OL-Rev GAT GTG CAC ATA ATA GAA ACT CAT
TCG AAA AGG GAA TGA T this study
P73 PciI-rzoR-rev CAC ATG TCC CAG TCG TTC CCG GAG GGT GAA PciI this study
P74 tonB-OL-Fw GTT TCT ATT ATG GTA CAT CAG GTT ATT GAA CTA CCT G this study
P75 tonB-OL-Rev GAT GTA CCA TAA TAG AAA CTC ATT CGA AAA GGG AAT GAT this study
P76 PciI-tonB-rev CAC ATG TC CTG AAT TTC GGT GGT GCC GTT PciI this study
P77 uvrB-OL-Fw GTT TCT ATT ATG AGT AAA CCG TTC AAA CTG AAT TCC G this study
P78 uvrB-OL-Rev GTT TAC TCA TAA TAG AAA CTC ATT CGA AAA GGG AAT GAT G this study
P79 PciI-uvrB-rev CAC ATG TCC GAT GCC GCG ATA AAC AGC TCA PciI this study
P80 hisD-TF-OL-Fw GTT TCT ATT ATG AGC TTT AAC ACA
ATC ATT GAC TGG AAT AG this study
P81 hisD-TF-OL-Rev TGT TAA AGC TCA TAA TAG AAA CTC
ATT CGA AAA GGG AAT GAT this study
P82 PciI-hisD-TF-rev CAC ATG TCT GCT TGC TCC TTA AGG
GCG TTA AC PciI this study
P83 flhC-TF-OL-Fw-APP GCG CGG AAG CGC ACC GCC GAA AG this study P84 flhC-TF-OL-Rev-APP CTT TCG GCG GTG CGC TTC CGC GC this study P85 flhC-TF-OL-FW-pap CTG CGC GGA AGC CCA GCG CCG AAA
GGC ATG this study
P86 flhC-TF-OL-rev-pap CAT GCC TTT CGG CGC TGG GCT TCC
GCG CAG this study
P87 flhC-TF-OL-FW-aaa CTG CGC GGA AGC GCA GCG GCG AAA GGC ATG this study
P88 flhC-TF-OL-rev-aaa CAT GCC TTT CGC CGC TGC GCT TCC GCG CAG this study
qRT-PCR-Primer
P89 Q-PCR-recA-Fw CGG TTC GCT TTC ACT GGA TAT CG this study P90 Q-PCR-recA-Rev CCT GCA GCG TCA GCG TGG T this study P91 Q-PCR-rpoD-Fw GCT GGC TGA AAA CAC CGC GG this study P92 Q-PCR-rpoD-Rev GCC CAT TTC ACG CAT GTA CAT GC this study P93 Q-PCR-cadC-Fw CGC AGA GTA TCT CAG AAC TAC GTA this study P94 Q-PCR-cadC-Rev CCT CTC CCT CTT CTT CGC TGT this study
P95 Q-PCR-cadA-Fw CGC GTT CGC TAA TAC GTA TTC CA this study P96 Q-PCR-cadA-Rev CGT CAG TGG TCT GCT TGA TCT TAT this study P97 Q-PCR-cadB-Fw CGA CTG GCA ACA AAA AAC CCG C this study P98 Q-PCR-cadB-Rev GCC AGG TTA CCA ATC CAG TTA GC this study
Primer deletion strains
P99 yjeK-mid-50
GAT TGT GAT CCC GGC ACG TAT CAC CGA GGC GCT GGT TGA ATG CTT TGC CCA ATT AAC CCT CAC TAA AGG GCG
(Kraxenberger, 2006)
P100 yjeK-down-50
AAG CGT AGC GAA TCA GGC AAT TTT AAT GTT TAA CTT CCC TGT TTA ATC AGT AAT ACG ACT CAC TAT AGG GCT C
![Figure 3: Translation initiation and elongation [reviewed in (Schmeing & Ramakrishnan, 2009)]](https://thumb-us.123doks.com/thumbv2/123dok_us/8069384.1345713/19.595.77.522.68.484/figure-translation-initiation-elongation-reviewed-schmeing-ramakrishnan.webp)
